Extended Data Figure 5: MNoV and HNoV NS3 N-terminal domain directly triggers cell death in multiple cell types.

NS3-FL, NS3-N or NS3-C fused with C-terminal Flag tag were stably expressed in BV2 cells under a tetracycline-inducible promoter. Protein expression was induced with doxycycline treatment. a, Representative bright-field images of BV2 cells stably expressing inducible constructs in the absence or presence doxycycline for 12 h. Scale bar, 100μm. b, Immunoblot of NS3 with anti-Flag antibody in the absence or presence doxycycline for 12 h. c, Cytotoxicity of BV2 cells expressing inducible constructs after doxycycline addition with or without 20 μM zVAD for 12 h. d, Representative Incucyte images of SytoxGreen-stained stably BV2 cells after doxycycline addition for 24 h. Scale bar, 200μm. e, BV2 cells expressing inducible constructs of NS3-FL, NS3-N and NS3-C. The time-course Incucyte quantification of SytoxGreen-positive cells in the absence or presence doxycycline. f, g, N-terminal Flag-tagged constructs encoding NS3-FL, NS3-N or NS3-C from MNoV^CR6 were transfected into A20 B cells (f) and HeLa cells (g). Cell death was determined by ATP-based cell viability assay 24 h after transfection. h, LDH released from HEK293T cells transfected with N-terminal Flag-tagged constructs encoding NS3-FL, NS3-N or NS3-C of HNoV MD145. i, Representative bright-field images of HEK293T cells transfected with N-terminal Flag-tagged constructs encoding NS3-FL, NS3-N or NS3-C of HNoV MD145. Scale bar, 100μm. j, The immunoblot shows expression of transfected FL-, N- or C-NS3 of MD145. Data in (a, b, d, e, i and j) are representative of three independent experiments. Data in (c, f, g and h) are pooled from three independent experiments with three technical replicates each. Data are presented as mean of biological replicates ± s.d.